The soybean cyst nematode (SCN), Heterodera glycines, is one of the most destructive plant-parasitic nematodes and has been found in most soybean-growing countries and regions in the world. The north central region of the United States is a major soybean-producing region and the nematode has been reported from all the states except North Dakota (Noel, 1992; Smolik, 1996). The nematode is a major yield-limiting factor of soybeans.
Management of the SCN has largely relied on rotation with non-host crops and planting resistant cultivars. In many cases, however, rotation and/or use of resistant cultivars are not efficient, or are impractical. Many factors including biotic and abiotic factors affect the efficacy of rotation and use of resistant cultivars. Results obtained in a recent crop rotation study indicated that 3 years of corn did not appear to be adequate to lower SCN density to a level where an SCN susceptible cultivar could be recommended in Minnesota. Further, increase of years of corn may result in corn yield penalty for the second and the following years for unknown reasons.
Use of resistant cultivars places a selection pressure on the nematode races. Continuous use of resistant cultivars with the same resistant source may result in race shift and eventually the resistance may be broken. Furthermore, the nematode can cause a significant yield loss even to resistant soybean cultivars. Therefore, it is important to reduce nematode density before planting a resistant cultivar.
Nematophagous fungi have been known for over 100 years and have been tested for biological control of plant parasitic nematodes for over 60 years (Linford, 1937; Zopf, 1888). Several fungi such as Arthrobotrys spp., Drechmeria coniospora (Drechsler) Gams and Jansson, Hirsutella rhossiliensis Minter and Brady, Paecilomyces lilacinus (Thom.) Samson, and Verticillium chlamydosporium Goddard, have been extensively studied but no successful biological control agents have been developed from these fungi (Galper et al., 1995; Stirling, 1991).
Nematophagous fungi have been isolated from various nematodes and locations. The fungi vary considerably among species and isolates in characteristics such as virulence to certain nematodes, colonization ability in plant roots, and competitive ability in soil (Boume et al., 1996; Timper and Riggs, 1998). The variability among isolates of H. rhossiliensis, P. lilacinus, and V. chlamydosporium has been demonstrated (Carneiro and Gomes, 1993; Irving and Kerry, 1987; Tedford et al., 1994). Waller and Faedo (1993) tested 94 species of nematode-trapping fungi for their infection of the free-living stage of animal-parasitic nematode, Haemonchus contortus Rudolphi in the sheep fecal environment and found only a few species with efficient activity.
Hirsutella rhossiliensis was first described in 1980 (Minter and Brady, 1980) based on a specimen collected from Wales in 1953. Sturhan and Schneider (1980) reported isolating this fungus from the hop cyst nematode, Heterodera humuli Filipjev, and named it Hirsutella heteroderae (synonym of H. rhossiliensis). The fungus has a wide range of hosts including plant-parasitic nematodes, free-living nematodes, entomopathogenic nematodes and mites, although different isolates may have different host preferences. Hirsutella rhossiliensis can parasitize a high percentage of nematodes in some locations. This fungus is probably an obligate parasite in nature and is generally isolated from only one species of nematode in a field (Jaffee and Zehr, 1985; Jaffee et al., 1991; Liu and Chen, 2000a; Sturhan and Schneider, 1980; Timper and Brodie, 1993; Velvis and Kamp, 1995).
Hirsutella rhossiliensis is a hyphomycetes with simple erect phialides which are swollen at the base and taper towards the apex. When a host nematode comes into contact with conidia on the phialides, the conidia can attach to the nematode cuticle, and infect the host nematode within a few days. Following penetration, the fungus forms an infection bulb in the nematode cavity, from which assimilative hyphae are developed. After converting nematode body contents to mycelial mass, the fungus may emerge from the nematode cadaver, produce spores, and infect other nematodes. An average of 112 conidia may be formed from mycelium developed from a single juvenile of H. schachtii at 20xc2x0 C. (Jaffee et al., 1990). KCl increased infection of nematodes by the fungus (Jaffee and Zehr, 1983). Conidia detached from the phialides may lose infectivity. Some conidia died shortly after sporulation and others may be viable and virulent for at least 200 days (Jaffee et al., 1990). Variability of morphology, pathogenicity, and genetics was observed among isolates (Tedford et al., 1994).
Parasitism of nematodes by H. rhossiliensis is dependent on nematode density. The percentage of nematodes parasitized by the fungus correlates positively with host nematode density (Jaffee et al., 1992). The number of conidia attached to cuticle of nematode by H. rhossiliensis correlates with the amount of conidia in the soil. Since the fungus is a poor soil competitor, local populations of the fungus may go extinct unless supplied with some minimum number of nematodes (the host threshold density). Thus, natural epidemics of this fungus among populations of nematodes develops slowly and only after long periods of high host densities (Jaffee and Zehr, 1985). Transmission of spores is greater in loamy sand than in coarse sand (Jaffee et al., 1990). In contrast to the theory that addition of organic matter may enhance activity of some nematophagous fungi, addition of organic matter to soil decreases parasitism of M. xenoplax by H. rhossiliensis (Jaffee et al., 1994).
Many endoparasitic nematophagous fungi produce adhesive spores, which adhere to passing vermiform nematodes, and subsequently infect, and kill the host. Drechmeria coniospora (Drechsler) Gams and Jansson (Drechsler, 1941), Hirsutella rhossiliensis Minter and Brady (Hirsutella heteroderae Sturhan and Schneider, Sturhan and Schneider 1980; Jaffee and Zehr, 1982), and Verticillium banaloides Drechsler (Drechsler, 1941) are well known species in this group. Only H. rhossiliensis, however, parasitizes high percentages of nematodes in natural soils. The fungus parasitized 80% of Mesocriconema xenoplax Raski in California peach orchard soils (Jaffee et al., 1988) and 90% of Heterodera schachtii Schmidt J2 in oil-radish fields in Germany (Mxc3xcller, 1982). Hirsutella rhossiliensis was naturally present in about 25% of the sugarbeet fields in Germany, in 17 of 20 fields in a starch-potato-growing area in the northeastern Netherlands (Velvis and Kamp, 1995), and in 10 of 21 sugarbeet fields in California, and may contribute to the suppression of H. schachtii (Mxc3xcller, 1984, 1986; Jaffee et al., 1991; Juhl, 1985). Jaffee and Muldoon (1989) also found that penetration of cabbage roots by H. schachtii was suppressed by 50-77% in loamy sand naturally infested with H. rhossiliensis. Hirsutella rhossiliensis has been isolated from Heterodera humuli Filipjev (Sturhan and Schneider, 1980), H. schachtii (Muller, 1984), Heterodera avenae Woll. (Stirling and Kerry, 1983), Heterodera glycines Ichinohe (Chen, 1997), Meloidogyne javanica (Treub) Chitwood (Cayrol et al., 1986), M. xenoplax (Jaffee and Zehr, 1982), Rotylenchus robustus (de Man) Filipjev (Jaffee et al., 1991), Xiphinema diversicacaudatum (Micoletzky) Thorne (Ciancio et al., 1986), Hoplolaimus galeatus Filip. and Schxc3xar. Stek., bacteria-feeding nematodes, soil mites, and soil (Tedford et al., 1994). It has also shown to infect Ditylenchus dipsaci (Kxc3xchn) Filipjev, Aphelenchoides fragariae (Ritz. Bos) Christie, Meloidogyne incognita (Kofoid and White) Chitwood (Cayrol and Frankowski, 1986; Cayrol et al., 1986), Pratylenchus penetrans (Cobb) Filip. and Schxc3xar. Stek (Timper and Brodie, 1993), Anguina sp. (Cayrol and Combettes, 1983), Anaplectus sp, Cephalobus (Sturhan and Schneider, 1980), and entomopathogenic species of Steinernema, Heterorhabditis (Timper et al., 1991) in laboratory and greenhouse studies.
The potential of the fungus as biological control agent has been controversial. Muller (1982) reported that the fungus might suppress cyst nematodes in some sugar beet fields in Germany. The fungus was considered to be partially responsible for suppression of M. xenoplax population in some orchards in the southern United States (Zehr, 1985). High numbers and percentages of M. xenoplax parasitized by H. rhossiliensis were also found in some California peach orchards (Jaffee et al., 1989). In greenhouse studies, H. rhossiliensis suppressed G. pallida on potato (Velvis and Kamp, 1996), H. schachtii on cabbage (Jaffee and Muldoon, 1989), and Pratylenchus penetrans Cobb on potato (Timper and Brodie, 1994).
Results obtained by Tedford et al. (1993), however, indicated that long-term interactions between populations of H. rhossiliensis and cyst or root-knot nematodes will not result in biological control. In a field microplot test, H. rhossiliensis failed in suppression of H. schachtii (Jaffee et al., 1996). H. rhossiliensis has been formulated in alginate pellets and used in control of nematodes in laboratory or greenhouse studies (Lackey et al., 1993; Jaffee et al., 1996). No commercial formulation, however, has yet been developed.
Biological control represents one of the components in an integrated pest management program and has been shown promise in control of many other agricultural pests. Especially in view of bans on chemical nematicides, such as the ban on methyl bromide, there remains a continuing need for a means to safely and effectively control the spread of nematodes, specifically of Heterodera glycines. Further, there is a long-felt, unresolved need to produce a pesticidal composition that can be sprayed, or similarly applied, onto crops to control nematodes.
The present invention provides a novel isolated and purified strain of fungus Hirsutella minnesotensis. The H. minnesotensis may be capable of controlling plant-parasitic nematodes, such as the nematode Heterodera glycines, and/or other agricultural pests. The H. minnesotensis may be culture deposit ATCC MYA-31 (CBS 102348) or any other isolate of the species such as isolates CBS 102457, CBS 102458 or CBS 102459.
The present invention also provides a pesticidal composition comprising an effective amount of an isolated and purified fungal strain of Hirsutella rhossiliensis or Hirsutella minnesotensis capable of controlling nematode infestation, and a carrier. The nematode may be a plant-parasitic nematode, such as Heterodera glycines, but is not limited to H. glycines. The Hirsutella rhossiliensis may be isolate OWVT-1, specifically culture deposit ATCC PTA-3179. which was deposited in the Amencan Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110 on Mar. 14. 2001. The H. minnesotensis may be culture deposit ATCC PTA-3180 (CBS 102348), which was denosited in the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110 on Mar. 14, 2001, or other isolate. The carrier may be diatomaceous earth, alginate, clay, or other plant and animal products as solid formulations. Alternatively the carrier may be a liquid form. An adjuvant or activator may be added into the solid or liquid formulations. Such an adjuvant or activator may increase dispersal, vegetative growth, sporulation, spore germination of the fungus, and/or inhibit soil microbial competitors. The activator may be in a monosaccharide, disaccharide, polysaceharide, amino acid, peptide, peptone, protein, vitamin, other organic compound, or inorganic salt. The Hirsutella strain may be in the spore and/or mycelium form. The pesticidal composition may contain an effective amount of at least one strain of Hirsutella rhossiliensis or Hirsutella minnesotensis that is in the range of about 1xc3x97102 to about 1xc3x971012 spores or colony forming units (cfu) per milliliter of liquid culture or per gram of solid culture. Alternatively, it maybe in the range of about 1xc3x97104 to about 1xc3x97109 spores or cfu per ml or gram, or even in the range of about 1xc3x97105 to about 1xc3x97108 spores or cfu per ml or gram.
The present invention further provides method for controlling nematodes comprising applying an effective amount of a pesticidal composition onto a target plant or onto the situs of a target plant (i.e., the area around the target plant), wherein the pesticidal composition comprises an effective amount of an isolated and purified fungal strain of Hirsutella rhossiliensis or Hirsutella minnesotensis capable of controlling nematode infestation and a carrier. The pesticidal composition is applied at least once, but may be applied a plurality of times in a single growing season, or over the course of several growing seasons.